Methods of producing bioconjugates of E. coli O-antigen polysaccharides, compositions thereof, and methods of use thereof

Patent metadata

Field	Value
Publication number	US-12233121-B2
Application number	US-202217938454-A
Country	US
Kind code	B2
Filing date	Oct 6, 2022
Priority date	Mar 18, 2019
Publication date	Feb 25, 2025
Grant date	Feb 25, 2025

Abstract

Methods of producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells are provided. The recombinant host cells used in the methods described herein encode a particular oligosaccharyl transferase enzyme depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase enzymes can be PglB oligosaccharyl transferase or variants thereof. Also provided are compositions containing the bioconjugates, and methods of using the bioconjugates and compositions described herein to vaccinate a subject against extra-intestinal pathogenic E. coli . (ExPEC).

First claim

The invention claimed is:

1. A recombinant host cell for preparing a bioconjugate of an E. coli Ox-antigen polysaccharide covalently linked to a carrier protein, the recombinant host cell comprising:
(a) a nucleotide sequence of an rfb gene cluster for the Ox-antigen polysaccharide;
(b) a nucleotide sequence encoding the carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1; and
(c) a nucleotide sequence encoding an oligosaccharyl transferase PglBy, wherein the PglBy comprises the amino acid substitution N311V relative to a wild-type PglB having the amino acid sequence of SEQ ID NO: 6,
wherein the E. coli Ox-antigen polysaccharide is the O1A antigen polysaccharide comprising the structure:

wherein n is independently an integer of 3 to 50.

2. The recombinant host cell of claim 1 , wherein
the PglB y comprises the amino acid mutations of N311V, K482R, D483H, and A669V relative to the wild-type PglB.

3. The recombinant host cell of claim 1 , wherein the host cell is a prokaryotic host cell.

4. The recombinant host cell of claim 3 , wherein the host cell is E. coli.

5. The recombinant host cell of claim 1 , wherein the carrier protein is selected from the group consisting of detoxified Exotoxin A of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), Diphtheria toxoid, Tetanus toxoid, detoxified hemolysin A of S. aureus , clumping factor A, clumping factor B, E. coli heat labile enterotoxin, detoxified E. coli heat labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified cholera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae Pneumolysin, Keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

6. The recombinant host cell of claim 1 , wherein the carrier protein comprises SEQ ID NO: 3.

7. The recombinant host cell of claim 1 , wherein the PglBy comprises the amino acid sequence of SEQ ID NO: 6 with the N311V substitution, the host cell is E. coli and the carrier protein comprises the amino acid sequence of SEQ ID NO: 3.

8. The recombinant host cell of claim 1 , wherein the PglBy comprises the amino acid sequence of SEQ ID NO: 6 with the N311V, K482R, D483H and A669V substitutions, the host cell is E. coli and the carrier protein comprises the amino acid sequence of SEQ ID NO: 3.

Assignees

• Janssen Pharmaceuticals Inc
• Glaxosmithkline Biologicals Sa
• Glaxosmith Kline Biologicals S A

Inventors

• Geurtsen Jeroen
• Burghout Pieter Jan
• Weerdenburg Eveline Marleen
• Poolman Jan Theunis
• Fae Kellen Cristhina
• Ibarra Yon Patricia
• Abbanat Darren Robert
• Kemmler Stefan Jochen
• Kowarik Michael Thomas
• Mally Manuela
• Gambillara Fonck Veronica
• Braun Martin Edward
• Carranza Sandmeier Maria Paula

Classifications

• C07K14/245

  Escherichia (G) · CPC title

• C07K14/21

  from Pseudomonadaceae (F) · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• A61K39/385

  Haptens or antigens, bound to carriers · CPC title